Method for detecting oxidized forms of soluble guanylate cyclase

ABSTRACT

The invention relates to methods for detecting a soluble guanylate cyclase whose heme iron is in the trivalent oxidation state, and to methods for finding chemical substances which stimulate the activity of a soluble guanylate cyclase when the heme iron of at least part of this soluble guanylate cyclase is oxidized to the trivalent state and also to diagnostic aids or kits for detecting a soluble guanylate cyclase with trivalent heme iron. Further, the invention relates methods for detecting a soluble guanylate cyclase lacking a heme group, and to methods for finding chemical substances which stimulate the activity of a soluble guanylate cyclase lacking a heme group.

The invention relates to methods for detecting soluble guanylate cyclasewhose heme-complexed iron is oxidized or which contains no heme group,to screening methods for identifying compounds able to activate solubleguanylate cyclase with oxidized heme iron, and to diagnostic aids fordetecting a soluble guanylate cyclase having a trivalent heme iron.

Soluble guanylate cyclases are heterodimeric proteins. They consist ineach case of an α subunit and a β subunit, and contain heme as aprosthetic group. Binding of the signal molecule nitric oxide (“NO”) tothe heme group activates the enzyme. The cGMP formed through theenzymatic activity of soluble guanylate cyclase is involved, inter alia,in the activation of cGMP-dependent protein kinases and in theregulation of phosphodiesterases or of ion channels. Four isoforms ofthe subunits have been described. These differ in their sequence and intheir tissue-specific and development-specific expression. Subtypes α₁and β₁ are found mainly in lung, kidney and brain. The β₂ chain isexpressed mainly in liver and kidney, and the α₂ subunit is expressedmainly in placenta. Dimerization of the subunits is a precondition for acatalytically active soluble guanylate cyclase. The heterodimers α₁/β₁,α₂/β₁, and α₁/β₂ are known. The region of the catalytic domain in allthe subunits shows a high degree of homology.

Soluble guanylate cyclase (sGC) contains one heme group per heterodimer.Binding of the heme takes place via His-105 in the β₁ chain. Mutants nolonger containing His-105 in the N terminus of the β₁ subunit cannot bestimulated by NO. The heme group in soluble guanylate cyclase consistsof an organic part of the molecule and an iron atom. The organic part,protoporphyrin IX, contains four pyrrole rings which are linked bymethine bridges to form a tetrapyrrole system. The iron atom in the hemegroup is bound to four nitrogen atoms in the center of theprotoporphyrin ring. In addition, it is able to engage in two otherlinkages. The oxidation state of the iron in the heme may be +2 (ferrousform) or +3 (ferric form; oxidized form). The oxidation state of theheme group iron has a crucial effect on the enzymatic function ofsoluble guanylate cyclase. The enzyme with a trivalent form heme ironshows only basal enzymatic activity, like a soluble guanylate cyclasewithout a heme group, and cannot be stimulated by NO. The preparation ofa heme-free soluble guanylate cyclase is described in Eur. J. Biochem.240, 380-386 (1996).

Soluble guanylate cyclase (sGC) catalyzes the conversion of GTP intocyclic guanosine monophosphate (cGMP) and pyrophosphate. cGMP acts as anintracellular messenger (second messenger). Second messengers areproduced inside the cell in cascade-like reactions. Their level iscontrolled by extracellular signals such as hormones, neurotransmitters,growth factors, odorous substances, peptides, or light. Formation ofsecond messengers serves to enhance signals. The second messengertransmits signals inside the cell to particular target proteins(kinases, phosphatases, ion channels, and others) which depend on thecell type. Modulation of soluble guanylate cyclase therefore leads, viathe influence on the cGMP levels and target proteins controlled thereby,to a number of pharmacological effects. Examples of mechanismsinfluenced in this way are the relaxation of smooth muscles (forexample, in the walls of blood vessels), the inhibition of plateletactivation, the inhibition of proliferation of smooth muscle cells, andthe adhesion of leukocytes.

Soluble guanylate cyclase is detectable in organs such as, for example,the heart, lung, liver, kidney, and brain of all mammals, includinghumans. In pathological processes or in processes relevant forpathological events, the oxidation state of the heme group iron insoluble guanylate cyclase may play an essential part. A higherproportion of soluble guanylate cyclase with oxidized heme group ironwould result in the possibility of diminishing activation of solubleguanylate cyclase by endogenous NO. This might lead, inter alia, to anincrease in blood pressure, activation of platelets, increasedproliferation of cells or enhanced adhesion of cells to permanent highblood pressure, stable or unstable angina pectoris, thromboses,myocardial infarct, strokes, pulmonary edemas, erectile dysfunction,uncontrolled tissue growth with tumor formation, diabetes, renaldysfunction, hepatic dysfunctions, or vascular dysfunction. Theendothelial cells of vessel walls secrete NO as paracrine hormone interalia for activating soluble guanylate cyclase. Compounds frequently usedfor pharmacological stimulation of soluble guanylate cyclase act as NOdonors via intermediate NO release. Examples of NO donors are theorganic nitrates. In addition, various compounds which do not act via NOrelease, but which modify the activity of soluble guanylate cyclase,have been described.

Activation of soluble guanylate cyclase by NO donors or free NO takesplace exclusively in the reduced, i.e., (Fe²⁺)-containing, state of hemeiron. This is evident from experiments carried out by A. Schrammel etal. in Mol. Pharmacol. 50, 1 (1996) with1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (“ODQ”). ODQ is a specificand highly effective inhibitor of soluble guanylate cyclase. ODQinteracts with the prosthetic heme group. In vitro, the substance causesirreversible oxidation of the heme iron of soluble guanylate cyclase.Treatment of soluble guanylate cyclase with ODQ results in thestimulating effect of NO on the enzyme being lost. Oxidation of the hemeiron in soluble guanylate cyclase can also be brought about withoxadiazolo(3,4-d)benz(b)(1,4)oxazin-1-one (Olesen et al., BritishJournal of Pharmacology 123, 299-309 (1998)) or potassium ferricyanide(Koesling et al., in “Reviews of Physiology Biochemistry andPharmacology,” pp. 41-65, Springer Verlag (1999)). There are alsoactivators of soluble guanylate cyclase which do not act via NO release.A description thereof has been given by, for example, Vesely et al. inEur. J. Clin. Invest. 15, 258 (1985). Stimulation of heme-free solubleguanylate cyclase by protoporphyrin IX has been demonstrated by Ignarroet al. in Adv. Pharmacol 26, 35 (1994). The effect of protoporphyrin IXcannot be inhibited by ODQ, but is instead enhanced (Koesling andFriebe, Physiol. Biochem. Pharmacol. 135, 41 (1999)). The activatorsdisclosed to date for soluble guanylate cyclase stimulate the enzymaticactivity thereof only if the heme iron is in the reduced, i.e.Fe²⁺-containing, state.

Recently, another class of chemical compounds has been described (WO00/02851), the sulfur-substituted sulfonylamino carboxylic acidN-arylamides, whose representatives are able to activate solubleguanylate cyclase.

The activity of soluble guanylate cyclase to date has been detected byenzymatic methods for detecting cGMP and cAMP, or by photometric methodsfor detecting the heme group. However, for establishing the dependenceof a condition with a pathological change on the state of solubleguanylate cyclase, it is insufficient merely to detect the protein byimmunological detection methods or by labeling techniques. It is just asimportant to know the redox state of the complexed iron in the hemegroup. Until now, it has only been possible to find information aboutthe functionality of the heme in soluble guanylate cyclase bydetermining the redox state of the complexed heme iron by ESRmeasurements or by photometry. These determinations have thedisadvantage that they depend on the availability of technically complexequipment. Moreover, the methods have only limited suitability forspecific measurements because impurities from other heme-containingproteins readily interfere.

One object of the present invention was therefore to provide simplifiedand specific methods for detecting an oxidation state of solubleguanylate cyclase. Another object of the invention was to providemethods for finding a chemical substance which activates solubleguanylate cyclase when its heme iron is oxidized to the trivalent state.The invention also relates to diagnostic aids which are suitable foridentifying soluble guanylate cyclases whose heme iron is oxidized inthe trivalent state.

The present invention relates to a method for detecting a solubleguanylate cyclase where the iron of the heme in this soluble guanylatecyclase is in the trivalent oxidation state, and where the methodcomprises the following steps:

a) provision of a soluble guanylate cyclase;

b) provision of at least one chemical compound which stimulates theactivity of a soluble guanylate cyclase when the iron of the heme inthis soluble guanylate cyclase is in the trivalent oxidation state;

c) incubation of a soluble guanylate cyclase provided as in a) with achemical compound provided as in b);

d) determination of the activity of soluble guanylate cyclase afterincubation as in c).

Specifically, the invention relates to a method for detecting solubleguanylate cyclase having a heme group with iron in a trivalent oxidationstate, comprising:

a) incubating a sample of soluble guanylate cyclase having a knownactivity with a chemical compound which stimulates activity of solubleguanylate cyclase having a heme group with iron in the trivalentoxidation state;

b) measuring the activity of the soluble guanylate cyclase afterincubation; and

c) comparing the activity of the soluble guanylate cyclase afterincubation with the activity before incubation, wherein an increase inactivity indicates the presence of soluble guanylate cyclase having aheme group with iron in the trivalent oxidation state.

The soluble guanylate cyclase may consist of a mixture of molecules ofsoluble guanylate cyclase, with not all the soluble guanylate cyclasemolecules being present with a heme iron in the trivalent oxidationstate. In particular, some of the heme groups of the soluble guanylatecyclase molecules can be in the trivalent oxidation state and someothers in the divalent oxidation state, to result in a mixture withvarying proportions of the oxidation states of the heme iron. The methodfor detecting a soluble guanylate cyclase whose heme iron is in thetrivalent form can be carried out by comparison with reference values.The reference values may be obtained inter alia from experiments inwhich the dependence of the activity of soluble guanylate cyclase onknown proportions of soluble guanylate cyclase with trivalent heme ironhas been determined. A measure of the particular proportion of solubleguanylate cyclase with trivalent heme iron can be obtained bypretreatment of the soluble guanylate cyclase with ODQ and subsequentdetermination of the activity of this soluble guanylate cyclase. Thedetermined reference values can then be used as calibration plots forquantitative determinations of soluble guanylate cyclase with heme ironin the trivalent oxidation state.

The soluble guanylate cyclase can be used in various states, for exampleas diverse heterodimeric soluble guanylate cyclase. It is possible inprinciple to employ a soluble guanylate cyclase from any species, inparticular, from any mammalian species. Soluble guanylate cyclase fromcattle is preferably used, and is preferably isolated from pulmonarytissue. Human soluble guanylate cyclase is particularly preferably used.The provided soluble guanylate cyclase can preferably be composed ineach case of an α subunit, for example of the α₁ or α₂ subunit, and of aβ subunit, for example of the β₁ or β₂ subunit. It is possible andpreferable for subunits of the soluble guanylate cyclase to be used forthe method of the present invention. Corresponding sequence informationis indicated in Giuili et al. (FEBS Letters 304, 83-88 (1992)) for thetwo subunits of soluble guanylate cyclase originally isolated from humanbrain, or in Nakane et al. (Journal of Biol. Chem. 265, 16841-16845(1990)) for cDNAs of the soluble guanylate cyclase from rat lung tissue,or in Yuen et al. (Biochemistry 29, 10872-10878 (1990)) for a solubleguanylate cyclase from rat kidneys.

The provision of a soluble guanylate cyclase can take place by isolationfrom a biological material using biochemical methods. In such cases inparticular there is frequently a mixture of soluble guanylate cyclasemolecules with different oxidation states of the heme iron. Biochemicalmethods may be, inter alia: methods for disintegrating the biologicalmaterial, centrifugation, chromatographic separations, gelelectrophoreses, isoelectric focusing, and immunological methods.Biological material may comprise eukaryotic cells or prokaryotic cells,disintegrates of the cells or preparations from disintegrates of thecells, whole cells or parts or fractions of the cells. The eukaryoticcells include inter alia, for example, cells from tissues or organs suchas heart, blood vessels, lung, blood, brain, liver, kidney, adiposetissue, muscle from vertebrates including humans, tissue or bloodsamples, but also cells from human or animal cell cultures, and insectcell cultures. Specific examples are platelets, which can be obtainedfrom human or animal blood samples, and smooth muscle cells, which canbe isolated, for example, from blood vessels of animal or human origin.Further examples are biopsy material, organs and tissues and partsthereof left over after transplantations, umbilical cords or placentae.Prokaryotic cells may be, for example, bacteria, including Escherichiacoli, Salmonella typhimurium, or Bacillus subtilis, as well as fungisuch as Saccharomyces cerevisiae, Saccharomyces pombe or Pichiapastoris. In a particular embodiment of the invention, the solubleguanylate cyclase can be recombinant material prepared, for example, byexpression in eukaryotic or prokaryotic cells, in which case the solubleguanylate cyclase, with or without a prosthetic heme group, isaccumulated within the cells or excreted into the medium, usingexpression vectors. In a specific embodiment, the soluble guanylatecyclase is isolated by a method as described by Humbert P. et al. inMethods of Enzymology 195, 384-391 (1991).

One embodiment provides a soluble guanylate cyclase which has beentreated with an oxidizing agent. Another embodiment provides a solubleguanylate cyclase which has been treated with ODQ. The concentration ofODQ is, for example, from 0.001 mM to 0.5 mM, preferably 0.005 mM to 0.1mM and, particularly preferably, 0.01 mM. It may also be preferred toprovide a soluble guanylate cyclase which has been treated withoxadiazolo(3,4-d)benz(b)(1,4)oxazin-1-one or another derivative of ODQ.In another preferred embodiment, a soluble guanylate cyclase which hasbeen treated with potassium ferricyanide is provided. The treatment ofthe soluble guanylate cyclase with oxidizing agents and/or ODQ and/orother compounds may cause oxidation of the heme iron, i.e., conversionof the iron from the divalent oxidation state (Fe²⁺) into the trivalentoxidation state (Fe²⁺). Depending on the chosen conditions, theconversion may affect only some or, in particular embodiments, all thetreated soluble guanylate cyclase molecules. The method of the presentinvention comprises using a form of soluble guanylate cyclase in whichthe heme iron is in the trivalent oxidation state either for only someof the soluble guanylate cyclase molecules or, in a preferredembodiment, in all of the soluble guanylate cyclase molecules.

The provision of the soluble guanylate cyclase comprises the preparationof suitable forms for incubation with selected chemical compounds, whereincubation means that the soluble guanylate cyclase and the chemicalcompound are brought into contact with one another. The protein can besuspended or dissolved, for example, in aqueous solvents supplemented bybuffers, ions, or else auxiliary reagents. It may also, for example, beattached to carrier material and used, immobilized in this form,suspended in solvents.

Chemical compounds which can be provided to stimulate the activity ofsoluble guanylate cyclase with trivalent heme iron may be eitherindividual chemical compounds or may be combinations of chemicalcompounds. The chemical compounds can be, for example, synthesized orisolated from natural substances. It is possible in principle to use forthe provision all chemical compounds which stimulate the activity of asoluble guanylate cyclase when the iron of the heme of this solubleguanylate cyclase is in the trivalent oxidation state.

To carry out the method for detecting soluble guanylate cyclase with aheme iron in the trivalent oxidation state it is possible to use, forexample, the chemical compound2-(4-chlorophenylsulfonylamino)-4,5-dimethoxy-N-(4-(thiomorpholine-4-sulfonyl)phenyl)benzamide.

To carry out the method for detecting a soluble guanylate cyclase with atrivalent heme iron, it is possible to use chemical compounds whichstimulate exclusively soluble guanylate cyclases with trivalent hemeiron. It is likewise possible to use compounds which, besides activationof the soluble guanylate cyclase with a trivalent heme iron, also showother effects on the soluble guanylate cyclase. For example, it ispossible to use chemical compounds which, besides activation of thesoluble guanylate cyclase with a trivalent heme iron, also causeactivation of the basal activity of the soluble guanylate cyclase oractivation of the soluble guanylate cyclase with a divalent heme iron. Amethod for detecting soluble guanylate cyclase using chemical compoundswhich activate exclusively the enzyme form with trivalent heme iron or,for example, also enzyme forms with divalent heme iron is carried outwith the assistance of reference values, calibration plots, and/or bycomparison with substances which are able to activate exclusively asoluble guanylate cyclase with divalent heme iron (for example NOdonors).

To construct a calibration plot, the soluble guanylate cyclase can beincubated with various concentrations of, for example, ODQ. Theparticular ratio of soluble guanylate cyclase with trivalent heme ironto soluble guanylate cyclase with divalent heme iron depends on the ODQconcentration employed in each case. The ODQ concentration used can beused directly as a measure of the proportion of enzyme with trivalentheme iron. The absolute proportion of soluble guanylate cyclase withtrivalent heme iron can be checked for calibration purposes using othermeasurement methods, for example by ESR measurements. The solubleguanylate cyclases pretreated differently with ODQ are activated by thechemical compounds in an incubation step. In this, compounds whichactivate exclusively a soluble guanylate cyclase with trivalent hemeiron give a value which is directly proportional to the amount ofsoluble guanylate cyclase with trivalent heme iron. Use of chemicalcompounds which, besides the soluble guanylate cyclase with trivalentheme iron, also activate the form with divalent heme iron, require anadditional calibration plot with a substance which activates a solubleguanylate cyclase exclusively in the divalent form. The proportion ofsoluble guanylate cyclase with trivalent heme iron then emerges bycomparing the activities obtained after different stimulation of asoluble guanylate cyclase pretreated with ODQ. For this, the solubleguanylate cyclase is stimulated once with a chemical compound whichactivates exclusively soluble guanylate cyclase with divalent heme ironand, on the other hand, a chemical compound which activates solubleguanylate cyclase with trivalent and divalent heme iron is used for thestimulation. Determination of the proportion of soluble guanylatecyclase with trivalent heme iron in a biological sample to beinvestigated results from comparison of the activity of the solubleguanylate cyclase from this sample after incubation with a chemicalcompound which is able to activate soluble guanylate cyclase asdescribed above with the values in a calibration plot.

The provision of a chemical compound may also comprise dissolving thiscompound in a suitable solvent or solvents, such as, for example,dimethyl sulfoxide (DMSO) or water, and preparation of a formulationwith auxiliary reagents.

The time for the incubation may vary in length, and various temperaturesmay be used. The time required and the temperature setting depend on theembodiments chosen in each case, considering the selection of thesoluble guanylate cyclase provided, the chemical compound provided,and/or other conditions such as the preparation form, the concentrationof the soluble guanylate cyclase employed and/or the chemical compoundemployed, and other additives. The incubation time can be, for example,between 1 minute and 120 minutes, a preferred incubation time is between1 minute and 60 minutes, and a time of 30 minutes is particularlypreferable. The temperature for the incubation can be between 5° C. and45° C., and the temperature is preferably between 20° C. and 42° C. and,in the particularly preferred embodiment, the temperature is 37° C.

The detection of the activity of the soluble guanylate cyclase can takeplace, for example, by an enzyme assay, by a subsequent functionalassay, or by a binding assay, for example in isolated cells or cellcultures. In a specific embodiment, this detection of the activity cantake place by finding the inhibition of platelet aggregation using aLumiaggregometer. Platelets aggregate less after activation of thesoluble guanylate cyclase and of the intracellular cGMP increase causedby the soluble guanylate cyclase. The extent of the inhibition ofplatelet aggregation is proportional to the activation of solubleguanylate cyclase and therefore provides a measure of the enzymeactivation. In this embodiment of the invention, the soluble guanylatecyclase is not provided in isolated form, but is present in theplatelet. The soluble guanylate cyclase can in principle also be used inthis form for the methods of the present invention, i.e., where thecyclase is present in whole cells. In another preferred embodiment, theactivity of soluble guanylate cyclase can also be measured bydetermining the cGMP formed using an RIA (radioimmunoassay) or EIA(enzyme immunoassay). In another preferred embodiment, the activity isdetermined via the effect of the chemical compounds on the cGMPformation in smooth muscle cells. Smooth muscle cells are a constituentof, for example, blood vessel walls. These muscle cells relax when theintracellular cGMP level increases. This may take place throughactivation of soluble guanylate cyclase. The relaxation of smooth musclecells can therefore be used to detect activators of soluble guanylatecyclase.

The active pharmaceutical ingredients disclosed to date cannot activatea soluble guanylate cyclase when its heme iron is in the trivalentstate. Chemical compounds able to activate soluble guanylate cyclasewhen its heme iron is oxidized in the tri-valent state have notpreviously been disclosed. Such chemical compounds could be used asactive pharmaceutical ingredients in pharmaceuticals for stimulatingsoluble guanylate cyclase, for example in a pathological state caused bya soluble guanylate cyclase with a trivalent heme iron. It wouldlikewise be possible to use such compounds in the method for detectingoxidized forms of soluble guanylate cyclase as described according tothe invention. Another embodiment of the invention therefore relates toa method for finding a chemical compound which stimulates the activityof a soluble guanylate cyclase when the iron of the heme of the solubleguanylate cyclase is in the trivalent oxidation state, the methodcomprising:

a) provision of a soluble guanylate cyclase where the iron of the hemeof this soluble guanylate cyclase is in the trivalent oxidation state;

b) provision of at least one chemical compound to be investigated;

c) incubation of a soluble guanylate cyclase according to a) with atleast one chemical compound to be investigated, according to b); and

d) determination of the activity of the soluble guanylate cyclase afterincubation as in c).

Therefore, the instant invention encompasses a method for identifying achemical compound which stimulates the activity of soluble guanylatecyclase having a heme group with iron in a trivalent oxidation state,comprising:

a) incubating a sample of soluble guanylate cyclase having a knownactivity and having a heme group with iron in the trivalent oxidationstate with a test chemical compound;

b) measuring the activity of soluble guanylate cyclase after incubation;and

c) comparing the activity of the soluble guanylate cyclase afterincubation with the activity before incubation, wherein an increase inactivity indicates the chemical compound stimulates the activity of thesoluble guanylate cyclase having a heme group with iron in the trivalentoxidation state.

A method for finding a chemical compound is also referred to asscreening. The use and provision of the soluble guanylate cyclase forthe screening method takes place as already stated for the method fordetecting a soluble guanylate cyclase with trivalent heme iron.

The provision of the soluble guanylate cyclase for the screening mayinclude the preparation of suitable forms for the incubation with achemical compound to be investigated. The soluble guanylate cyclase can,for example, be suspended or dissolved in aqueous solvents supplementedby buffers, ions, and/or auxiliary reagents. It can also be attached tocarrier material and can be used immobilized in this form suspended insolvents.

The chemical compound to be investigated in the screening may be achemically synthesized compound, or a natural product, or a combinationthereof. The chemical compound to be investigated may be present invarious states of matter, for example, as a single substance in purifiedform, as mixture with other compounds which need not be characterized indetail, as mixture of various active compounds, as constituent of amixture of compounds of biological origin, or together with eukaryoticand/or prokaryotic organisms.

The screening method can be carried out, for example, by a laboratoryrobot or with the assistance of machines. Chemical compounds to beinvestigated, which activate a soluble guanylate cyclase with atrivalent heme iron, may be used as active pharmaceutical ingredientsfor treating conditions with pathological changes, such as, for example,cancer, angina pectoris, diabetes, myocardial infarct, erectiledysfunction, heart failure, high blood pressure, thromboses, anemia, orvascular dysfunction. The invention also relates to the chemicalcompound to be investigated which is identified by this screening methodas activator of a soluble guanylate cyclase whose heme iron is in thetrivalent oxidation state.

The invention relates very generally to compounds which are able toactivate the activity of a soluble guanylate cyclase whose heme iron isin the trivalent oxidation state, and to the use of these substances,preferably as active pharmaceutical ingredients. The inventionaccordingly also relates to compounds which are able to activate asoluble guanylate cyclase whose heme iron is in the trivalent oxidationstate, and which can therefore be employed as active pharmaceuticalingredients for the treatment or prevention of pathological statescaused by or intensified by soluble guanylate cyclase with heme iron inthe trivalent oxidation state. Such pathological states may include, forexample, cancer, angina pectoris, diabetes, myocardial infarct, erectiledysfunction, heart failure, high blood pressure, thromboses, vasculardysfunction, or anemia.

The invention also relates to a diagnostic aid or kit for carrying out amethod for determining the oxidation state of heme iron in a solubleguanylate cyclase. The diagnostic aid can for this purpose comprise atleast one or more of the following components: a) at least one chemicalcompound which stimulates the activity of soluble guanylate cyclase whenthe heme iron of this soluble guanylate cyclase is in the trivalentoxidation state, and b) solutions and/or reagents for determining theactivity of a soluble guanylate cyclase. An example of a chemicalcompound which can be used in the diagnostic aid or kit is2-(4-chlorophenylsulfonylamino)-4,5-dimethoxy-N-(4-(thiomorpholine-4-sulfonyl)phenyl)benzamide.The activity can be determined by one of the methods already describedabove.

The diagnostic aid may also comprise, for example, soluble guanylatecyclase, a chemical compound which activates a soluble guanylate cyclaseonly when the heme iron is in the divalent state, an oxidizing agent,and/or ODQ. These constituents can be used singly or in combination toestablish a reference value or a calibration plot for quantitative orqualitative determination of soluble guanylate cyclase with a heme ironin the trivalent oxidation state.

The diagnostic aid may be used, for example, for determining the contentof soluble guanylate cyclase with trivalent heme iron in a biologicalsample. A biological sample might consist, for example, of cells from atissue or organ of a vertebrate or of a human. These cells might havebeen taken from an organism or have been grown by cell culturingtechniques. The donor organisms which may be selected are individualswith tissues or organs which are healthy or show pathological changes.For example, organ samples, tissue samples, blood samples, cells orbiopsy material can be taken using medical techniques from human oranimal organisms and serve as biological sample. It is also possible tocarry out comparative analysis of the biological samples from differentindividuals in connection with the oxidation state of the heme iron inthe soluble guanylate cyclase in the light of their different living andeating habits. Thus, for example, the cells of smokers and nonsmokerscould be obtained and analyzed. The soluble guanylate cyclases of thesmokers could be compared with those of nonsmokers for differences inthe ratio of divalent heme iron to trivalent heme iron.

The invention relates in a preferred embodiment to an in vitro methodfor determining the oxidation state of the heme iron in a solubleguanylate cyclase in a biological sample which has been taken from aeukaryotic organism. The biological sample taken from a eukaryoticorganism may contain, for example, healthy or pathologically changedcells from cell cultures, tissues, or organs.

In another preferred embodiment, a diagnostic aid described according tothe invention can be used to carry out the in vitro method. Examples ofpossible biological states with pathological changes are cancer, anginapectoris, diabetes, erectile dysfunction, myocardial infarct, heartfailure, high blood pressure, thromboses, anemia, vascular dysfunction,and others. It is also possible to investigate certain living or eatinghabits such as, for example, smoking or alcohol consumption for theeffects on the oxidation state of heme iron in the soluble guanylatecyclase.

In healthy biological states, the parameters used for thecharacterization fall within the normal range. Appropriate informationfor establishing the normal ranges is to be found, for example, intextbooks of clinical chemistry (for example Clinical Chemistry,Principles, Procedures, Correlations, Michael L. Bishop and Janet L.Duben-Enkelkirk, Eds., Lippincott, Williams & Williams).

The invention also relates to a method for detecting a soluble guanylatecyclase using a luminometric assay. The assay comprises several steps,carried out successively or simultaneously. The luminometric assaycomprises the following steps: a) provision of a soluble guanylatecyclase and GTP as substrate, b) conversion of the GTP by the providedsoluble guanylate cyclase to form cGMP and pyrophosphate, c) provisionof a nicotinamide mononucleotide adenyltransferase and nicotinamidedinucleotide (NAD⁺), d) conversion of the pyrophosphate formed in b) bythe nicotinamide mononucleotide adenyltransferase in the presence ofnicotinamide dinucleotide (NAD⁺) to form ATP, e) provision of aluciferase and its substrate luciferin, and f) luminometricdetermination of the formed ATP by reaction with luciferin underluciferase catalysis. The amount of formed ATP is proportional to theconcentration of soluble guanylate cyclase. The method is started byadding soluble guanylate cyclase to a reaction mixture which, besidesother substances, contains GTP as substrate. Other substances presentcan be, for example, buffers, ions, or proteins.

The method for detecting the soluble guanylate cyclase is suitable in apreferred embodiment for carrying out a screening of chemical compoundsto be investigated for their suitability for stimulating the activity ofsoluble guanylate cyclase. The advantage of this luminometric methodcompared with methods previously used for detecting soluble guanylatecyclase (i.e., immunological, enzymatic, or photometric methods) lies inthe ability to screen large quantities of chemical compounds for theirpotential ability to stimulate the activity of a soluble guanylatecyclase with trivalent heme iron in considerably shorter times. To carryout the screening, the chemical compound to be investigated is added tothe reaction mixture before the start of the method by addition ofsoluble guanylate cyclase. The method is suitable in a particularembodiment of the screening for use in a laboratory robot, and inanother particularly preferred embodiment for manual performance. Theprovision of the soluble guanylate cyclase can take place as alreadystated in the previous sections for the soluble guanylate cyclase. Aparticular advantage of this luminometric assay is that the componentsfor carrying out the method, such as GTP, nicotinamide mononucleotideadenyltransferase, NAD⁺, luciferin or luciferase, can be provided in areaction vessel. The activity of soluble guanylate cyclase isestablished by determining the amount of ATP formed. The reaction isstarted by adding soluble guanylate cyclase. This makes it possible tocarry out the method in a one-pot reaction. This can also be applied toscreening with high throughput of chemical substances to be investigated(high throughput screening; HTS) and can be carried out reproducibly.This luminometric assay is very suitable in particular for detectingsmall amounts of pyrophosphate. The method can in a preferred embodimentbe used to determine the activity of a soluble guanylate cyclase. Theluminometric assay is also suitable for carrying out a method asdescribed in the preceding sections for detecting soluble guanylatecyclase with trivalent heme iron.

The invention also relates to a method for detecting a soluble guanylatecyclase where the soluble guanylate cyclase has no heme group. Themethod comprises: a) provision of a soluble guanylate cyclase without aheme group, b) provision of at least one chemical compound able tostimulate the activity of soluble guanylate cyclase when the solubleguanylate cyclase has no heme group, c) incubation of a solubleguanylate cyclase with a chemical compound able to stimulate theactivity of a soluble guanylate cyclase without a heme group and d)determination of the activity of the soluble guanylate cyclase without aheme group after incubation with the chemical compound.

The invention also relates to a method for finding a chemical compoundwhich stimulates the activity of a soluble guanylate cyclase where thesoluble guanylate cyclase has no heme group. This method can comprise assteps a) provision of a soluble guanylate cyclase which has no hemegroup, b) provision of at least one chemical compound to beinvestigated, c) incubation of a soluble guanylate cyclase without hemegroup with at least one chemical compound to be investigated, and d)determination of the activity of the soluble guanylate cyclase afterincubation.

The invention also relates to a diagnostic aid for determining theoxidation state of a soluble guanylate cyclase without a heme group,comprising at least one chemical compound which stimulates the activityof a soluble guanylate cyclase where the soluble guanylate cyclase hasno heme group. The invention also relates to an in vitro method fordetermining a soluble guanylate cyclase which has no heme group in abiological sample, where the method may comprise as steps the provisionof a biological sample which contains a soluble guanylate cyclasewithout heme group, the provision of at least one chemical compound ableto stimulate the activity of a soluble guanylate cyclase without a hemegroup, the incubation of a soluble guanylate cyclase without heme groupwith a chemical compound able to stimulate the activity of a solubleguanylate cyclase, and the determination of the activity of the solubleguanylate cyclase without heme group after incubation with the chemicalcompound. A method for provision of a soluble guanylate cyclase withouta heme group is described in Foerster, J. et al., Eur. J. Biochem. 240,380-386 (1996). An example of a suitable chemical compound forstimulating a soluble guanylate cyclase without a heme group is thechemical compound2-(4-chlorophenylsulfonylamino)-4,5-dimethoxy-N-(4-thiomorpholine-4-sulfonyl)phenyl)benzamide.

EXAMPLES Example 1

Luminometric Method for Detecting the Activity of Soluble GuanylateCyclase

Soluble guanylate cyclase (sGC) catalyzes the conversion of GTP intocGMP and pyrophosphate. In the presence of nicotinamide adeninedinucleotide (NAD⁺) and nicotinamide mononucleotide adenylyltransferase(NAT, EC 2.7.7.1), pyrophosphate is converted into ATP and nicotinamidemononucleotide. The ATP formed can be quantified luminometrically withthe aid of a luciferase in microtiter plates.

The substance to be investigated is dissolved in DMSO and diluted withDMSO/water to a final concentration of 50 μM in the assay mixture. TheDMSO concentration in the assay mixture should not exceed 5% (v/v).

100 μl of reaction mixture should contain 50 mM TEA buffer (pH 7.4), 3mM MgCl₂, 3 mM GSH, 0.1 mM GTP, 1 mM IBMX (isobutylmethylxanthine), 0.2mM NAD⁺, 0.4 mU of NAT, suitably diluted sGC enzyme solution (isolatedfrom bovine lung as described by P. Humbert et al., Methods ofEnzymology 195, 384-391 (1991)) and the test substance or solvent (fordetermining the basal enzyme activity). The reaction is started byadding the sGC. The reaction mixture is incubated at room temperaturefor 60 min and then stopped by cooling in ice and adding 50 mM EDTA, pH8.0. For the ATP determination, 20 μl of 100 mM MgCl₂ and 50 μl of ATPassay reagent (0.035 mM luciferin and 10,000 U/mL luciferase in 62.5 mMTRIS acetate pH 7.75, 1.9 mM EDTA, 0.05 mM DTT, 0.1% BSA) are added. Therelative luminescence units (RLU) can be measured in a microtiter plateluminometer.

The sGC activity is obtained after subtracting the blank RLU (incubationwithout enzyme) from the measured RLU. Activation of the sGC by a testsubstance is indicated as a percentage of the basal enzyme activity(solvent control) and is calculated by the following formula:

100×(DRLU_(test substance)/DRLU_(control))−100=% activation

Example 2

Activation of Soluble Guanylate Cyclase after Oxidation of the Heme Iron

Isolated sGC is present in solution in a heme Fe²⁺-containing (reduced)form which can be stimulated by NO and a heme Fe³⁺-containing (oxidized)form which cannot be stimulated by NO. The proportion of each formpreviously was determined only by ESR measurements.

Nitric oxide (NO) and NO donors activate exclusively the reduced stateof sGC. On incubation in the presence of, for example, 0.01 mM1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ), which irreversiblyoxidizes sGC in vitro, they show complete loss of their stimulatingeffect. A novel NO-independent activator of sGC,1-benzyl-3-(2-hydroxymethyl-5-furyl)indazole (YC-1), likewise activatesthe reduced form of sGC and can be inhibited by ODQ.

In contrast, stimulation by substances which activate exclusively orpredominantly the oxidized form is either enhanced or unaffected by ODQ,depending on the proportion of the oxidized form in the mixture.

These substances likewise frequently show an activation of the heme-freeform of sGC. The preparation of a heme-free sGC is carried out accordingto J. Foerster et al., Eur. J. Biochem. 240, 380-386 (1996).

A soluble guanylate cyclase with a trivalent heme iron was prepared bystoring freshly prepared enzyme at 4° C. for about 7 days. Incubation ofsoluble guanylate cyclase pretreated in this way with2-(4-chlorophenylsulfonylamino)-4,5-dimethoxy-N-(4-(thiomorpholine-4-sulfonyl)phenyl)benzamideresulted in 17.6-fold stimulation of the activity. The EC50 was 0.5μmol/l. The stimulation on use of a heme-free soluble guanylate cyclasereached 58.2-fold. The affinity in this case was 2.4 μmol/l.

Example 3

Detection of the Stimulation of Soluble Guanylate Cyclase afterOxidation of the Heme Iron by Determining the Effect on PlateletAggregation

The physiological effect of nitric oxide (NO) such as inhibition ofplatelet aggregation and relaxation of smooth vascular muscles is theconsequence of direct activation of soluble (reduced) guanylate cyclaseby NO with increased formation of cGMP.

ODQ, a selective sGC inhibitor, inhibited, through oxidation of the sGC,the increase in cGMP and the antiaggregatory effect of NO donors inwashed human platelets (M. A. Moro et al., PNAS 93, 1480-1485 (1996)).

To prepare washed human platelets (WP), blood is taken from theantecubital vein and anticoagulated in the syringe with citricacid/sodium citrate. After centrifugation at 16×g for 15 min, thesupernatant consists of platelet-rich plasma (PRP). The PRP isacidified, and the platelets are sedimented by centrifugation at 400×gfor 20 min and taken up in Tyrode solution. These washed platelets (WP,3×10⁵/μL) are employed in the tests.

The inhibition of aggregation by sGC activators is determined in aLumiaggregometer. The WPs are incubated with the test substance in thepresence of 0.5 mM CaCl₂ at 37° C., and the reaction is started byadding, for example, 0.3 μg/mL collagen. The effect of the substances onaggregation is investigated in the absence and presence of ODQ. sGCactivators will show dose-dependent inhibition of collagen-inducedplatelet aggregation. Activators which stimulate the oxidized form ofsGC will show a stronger antiaggregatory effect in the presence of ODQ.The IC₅₀ for the inhibition of aggregation will be shifted to lowerconcentrations.

To determine the intracellular cGMP concentration, WPs are incubated inthe presence of 0.1 mM isobutylmethylxanthine (IBMX) at 37° C. for 15min. The incubation is stopped by centrifugation, and the precipitate istreated with 1M perchloric acid and ultrasound for 1 min. After renewedcentrifugation at 13,000×g for 15 min, the supernatant is neutralizedwith 1M KOH, and the cGMP concentration is determined using a commercialenzyme immunoassay (available from Amersham, inter alia) for cGMP. Theprotein concentration in the precipitate is determined by the Bradfordmethod.

Chemical compounds which activate the activity of soluble guanylatecyclase will lead to a concentration-dependent increase in theintracellular cGMP concentration. If the incubation is carried out inthe presence of ODQ, it should be possible to obtain significantlyhigher cGMP levels than without ODQ using chemical compounds whichstimulate the activity of soluble guanylate cyclase with trivalent hemeiron, i.e., ODQ should enhance the effect of the substances.

Example 4

Detection of the Stimulation of Soluble Guanylate Cyclase afterOxidation of the Heme Iron by Determining the Effect on Smooth MuscleCells from Rat Aorta

Smooth muscle cells (VSMC) from rat aorta are isolated and cultivated bythe method of J. H. Chamley et al., Cell Tissue Res. 177, 503-522(1977).

The cells are seeded in 6-well plates and incubated in HEPES-Tyrodebuffer (pH 7.4 with 200 U SOD, 0.3 mM IBMX) at 37° C. for 15 min. Theeffect of the substances on the VSMC is investigated in the absence andpresence of ODQ. The incubation is stopped by aspirating off thesupernatant and adding liquid nitrogen, and the cells are deep-frozen inthe 6-well plates. To determine cGMP, the plates are thawed, theindividual wells are charged with buffer, and the cGMP concentration inthe supernatant is determined with a commercial enzyme immunoassay(available from Amersham, inter alia) for cGMP. The proteinconcentration is determined by the Bradford method after the protein inthe wells has been dissolved with 0.1 M NaOH.

Chemical compounds which stimulate the activity of soluble guanylatecyclase will lead to a concentration-dependent increase in theintracellular cGMP concentration. If the incubation is carried out inthe presence of ODQ considerably higher cGMP levels will be obtainedwith chemical compounds which stimulate the activity of solubleguanylate cyclase with a trivalent heme iron. ODQ ought to enhance theeffect of the activating compounds.

Example 5 Preparation of2-(4-chlorophenylsulfonylamino)-4,5-dimethoxy-N-(4-(thiomorpholine-4-sulfonyl)phenyl)benzamide

33.71 g (0.32 mol) of sodium carbonate were dissolved in 250 ml of waterand heated to 60° C. 25.00 g (0.13 mol) of 2-amino-4,5-dimethoxybenzoicacid were introduced into the solution, and 29.55 g (0.14 mol) of4-chlorobenzenesulfonyl chloride were added in portions to this solutionover the course of 15 min. After the mixture had been cooled, theresidue was filtered off with suction and taken up in 1% strength sodiumbicarbonate solution and, after filtration, the product was precipitatedby adding 1 N hydrochloric acid. 25.90 g (55%) of2-(4-chlorophenylsulfonylamino)-4,5-dimethoxybenzoic acid of meltingpoint 212-214° C. were obtained. 25.90 g (0.07 mol) of2-(4-chlorophenylsulfonylamino)-4,5-dimethoxybenzoic acid were suspendedin 75 ml of toluene and, after addition of 17.30 g (0.08 mol) ofphosphorus pentachloride, the mixture was stirred at 40-45° C. for 2.5h. It was then concentrated to half the volume in vacuo, and theprecipitated product was filtered off with suction and washed with alittle toluene. 25.30 g (93%) of2-(4-chlorophenylsulfonylamino)-4,5-dimethoxybenzoyl chloride, ofmelting point 175-177° C., were obtained.

10.00 g (25.6 mmol) of2-(4-chlorophenylsulfonylamino)-4,5-dimethoxybenzoyl chloride weresuspended in 300 ml of toluene, 4.49 g (25.6 mmol) of4-aminobenzenesulfonyl fluoride were added, and the mixture was heatedunder reflux for 4 h. After cooling, the precipitate which had separatedout was filtered off with suction and washed with toluene. 11.71 g (87%)of the title compound, of melting point 216-219° C., were obtained.

500 mg (0.95 mmol) of4-((2-(4-chlorophenylsulfonylamino)-4,5-dimethoxybenzoyl)amino)benzenesulfonylfluoride were dissolved in 1 ml of thiomorpholine and heated at 90° C.for 30 min. For workup, the mixture was poured into 50 ml of ice/1 Nhydrochloric acid, and the precipitate was filtered off with suction,dried in a vacuum dryer over phosphorus pentoxide, and recrystallizedfrom hexane/ethyl acetate. 378 mg (65%) of the title compound of meltingpoint 241° C. were obtained.

The present invention may be embodied in other specific forms withoutdeparting from its spirit or essential characteristics. The describedembodiments are to be considered in all respects as illustrative onlyand not restrictive. The scope of the invention is, therefore, indicatedby the appended claims rather than by the foregoing description. Allchanges which come within the meaning and range of equivalency of theclaims are to be embraced within their scope.

We claim:
 1. A method for detecting soluble guanylate cyclase having aheme group with iron in a trivalent oxidation state, comprising: a)incubating a sample of soluble guanylate cyclase having a known activitywith a chemical compound which stimulates activity of soluble guanyl atecyclase having a heme group with iron in the trivalent oxidation state;b) measuring the activity of the soluble guanylate cyclase afterincubation; and c) comparing the activity of the soluble guanylatecyclase after incubation with the activity before incubation, wherein anincrease in activity indicates the presence of soluble guanylate cyclasehaving a heme group with iron in the trivalent oxidation state.
 2. Themethod as claimed in claim 1, wherein the sample of soluble guanylatecyclase is from a mammal.
 3. The method as claimed in claim 2, whereinthe sample of soluble guanylate cyclase is from cattle.
 4. The method asclaimed in claim 2, wherein the sample of soluble guanylate cyclase isfrom a human.
 5. The method as claimed in claim 1, wherein the sample ofsoluble guanylate cyclase comprises an α subunit and a β subunit,wherein the α subunit is an α₁ or α₂ subunit and the β subunit is a β₁or β₂ subunit.
 6. The method as claimed in claim 1, wherein the sampleof soluble guanylate cyclase is isolated from a biological material. 7.The method as claimed in claim 1, wherein the activity of the solubleguanylate cyclase is determined by a functional assay or a bindingassay.
 8. The method as claimed in claim 1, wherein the sample ofsoluble guanylate cyclase is treated with an oxidizing agent.
 9. Themethod as claimed in claim 8, wherein the sample of soluble guanylatecyclase is treated with 1H-(1,2,4)oxadiazolo(4,3-a)quinoxalin-1-one. 10.The method as claimed in claim 8, wherein the sample of solubleguanylate cyclase is treated withoxadiazolo(3,4-d)benz(b)(1,4)oxazin-1-one.
 11. The method as claimed inclaim 8, wherein the sample of soluble guanylate cyclase is treated withpotassium ferricyanide.
 12. A method for detecting soluble guanylatecyclase lacking a heme group, comprising: a) incubating a sample ofsoluble guanylate cyclase having a known activity with a chemicalcompound which stimulates the activity of a soluble guanylate cyclaselacking a heme group; b) measuring the activity of the sample afterincubation; and c) comparing the activity of the sample after incubationwith the activity before incubation, wherein an increase in activityindicates the presence of the soluble guanylate cyclase lacking a hemegroup.